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Downregulation of N-terminal initiator methionine imprinting by NatB impacts growth and the response to osmotic stress in Arabidopsis

ABSTRACT: N-terminal acetylation (NTA) is one of the most abundant protein modifications in eukaryotes and is catalyzed in humans by seven Nα-acetyltransferases (NatA-F and NatH). Remarkably, the characterization of the plant Nat machinery and its biological relevance is still in its infancy, although NTA has gained recognition as key regulator of crucial processes like protein turnover, protein-protein interaction and protein targeting. In this study we combined in vitro assays, reverse genetics, quantitative N-terminomics, transcriptomics and physiological assays to characterize the Arabidopsis NatB complex. We show that the plant NatB catalytic (NAA20) and auxiliary subunit (NAA25) form a stable heterodimeric complex that accepts canonical NatB-type substrates in vitro. In planta, NatB complex formation was essential for enzymatic activity. Depletion of NatB subunits to 30% of wild-type level in three Arabidopsis T-DNA insertion mutants (naa20-1, naa20-2, naa25-1) decreased growth to 50% of wild-type level. A complementation approach revealed functional conservation between plant and human catalytic NatB subunits, while yeast NAA20 failed to complement naa20-1. Quantitative N-terminomics of approximately 2000 peptides identified 29 bona fide substrates of the plant NatB. In vivo, NatB preferentially acetylated N-termini starting with the initiator methionine followed by acidic amino acids and contributed 20% of the acetylation marks in the detected plant proteome. The global transcriptome and proteome analyses of NatB-depleted mutants suggested a function of NatB in multiple stress responses. In agreement, we revealed the specific impact of NatB on the resistance of plants to osmotic or high-salt stress. Remarkably, depletion of NatA did not affect these resistances.

OTHER RELATED OMICS DATASETS IN: PXD016209

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE132978 | GEO | 2020/04/14

REPOSITORIES: GEO

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Project description:In Arabidopsis thaliana the evolutionary conserved N-terminal acetyltransferase (Nat) complexes NatA and NatB co-translationally acetylate 60% of the proteome. Both have recently been implicated in the regulation of plant stress responses. While NatA mediates drought tolerance, NatB is required for pathogen resistance and the adaptation to high salinity and high osmolarity. Salt and osmotic stress impair protein folding and result in the accumulation of misfolded proteins in the endoplasmic reticulum (ER). The ER-membrane resident E3 ubiquitin ligase DOA10 targets misfolded proteins for degradation during ER stress and is conserved among eukaryotes. In yeast, DOA10 recognizes conditional degradation signals (Ac/N-degrons) created by NatA and NatB. Assuming that this mechanism is preserved in plants, the lack of Ac/N-degrons required for efficient removal of misfolded proteins might explain the sensitivity of NatB mutants to protein harming conditions. In this study, we investigate the response of NatB mutants to dithiothreitol (DTT) and tunicamycin (TM) induced ER stress. We report that NatB mutants are hypersensitive to DTT but not TM, suggesting that the DTT hypersensitivity is caused by an over-reduction of the cytosol rather than an accumulation of unfolded proteins in the ER. In line with this hypothesis, the cytosol of NatB depleted plants is constitutively over-reduced and a global transcriptome analysis reveals that their reductive stress response is permanently activated. Moreover, we demonstrate that doa10 mutants are susceptible to neither DTT nor TM, ruling out a substantial role of DOA10 in ER-associated protein degradation (ERAD) in plants. Contrary to previous findings in yeast, our data indicates that N-terminal acetylation (NTA) does not inhibit ER targeting of a substantial amount of proteins in plants. In summary, we provide further evidence that NatB-mediated imprinting of the proteome is vital for the response to protein-harming stress and rule out DOA10 as the sole recognin for substrates in the plant ERAD pathway.

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Downregulation of N-terminal initiator methionine imprinting by NatB impacts growth and the response to osmotic stress in Arabidopsis

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